Functional Analysis of Endothelin Receptor Signaling in Zebrafish Enteric Nervous System Development 公开
Wang, Sijia (2014)
Published
Abstract
Endothelin-3 (EDN3) and its obligate receptor endothelin receptor B (EDNRB) are thought to play an essential role in enteric nervous system (ENS) development by regulating the proliferation and differentiation of enteric neural crest cells and their precursors. Patients with pediatric condition Hirschsprung disease (HSCR), a congenital ENS disorder, have been identified with a missense point mutation in the human EDNRB gene. To confirm the evolutionary conservation for the requirement of Endothelin-3 signaling in ENS development, we have identified and cloned two EDNRB orthologues in zebrafish. Bioinformatics analysis has revealed that the second copy of EDNRB gene has arisen because of the hypothesized genome duplication that occurred in teleosts after they diverged from the other vertebrate lineages. Subsequently, antisense digoxigenin-labeled probes were generated to characterize the expression pattern of EDNRB orthologues during embryogenesis. We found that both EDNRB1a and EDNRB1b orthologues are expressed in the neural crest precursors as these cells begin to enter the gastrointestinal tract and migrate caudally along its length. These results support our hypothesis that there is a conserved function of EDNRB orthologues in neural crest migration and ENS development in zebrafish model system.
Table of Contents
Introduction ........................................................................ 1
Materials and Methods ..........................................................4
Bioinformatics analysis .............................................................4
PCR cloning and sequencing ......................................................4
Whole-mount in situ hybridization ..............................................5
EDNRB1a and EDNRB1b antisense oligo injections ........................5
Results ..................................................................................7
Evolutionary conservation of EDNRB1a and EDNRB1b ...................7
Likely occurrence of a genome duplication event ..........................8
Expression of EDNRB orthlogues in the neural crest cells ...............9
Discussion ............................................................................10
References ...........................................................................12
List of Figures
Figure 1 Gene comparison between EDNRB1a and EDNRB1b .........15
Figure 2 EDNRB homolog analysis between different species .........16
Figure 3 Alignment of the amino acid sequences ..........................17
Figure 4 Syntenic analysis of the EDNRB orthologues ...................18
Figure 5 Rooted phylogram of the EDNRB orthologue family ..........19
Figure 6 Gel image of the DIG-labeled riboprobes .........................20
Figure 7 EDNRB1a and EDNRB1b spatial and temporal expression ...21
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